This invention relates to quantitative analysis of chromatographic effluents containing biomarkers symptomatic of disease states and tissue injury in humans.
Biomarkers of interest are proteins or isoenzymes (enzymes which catalyze the same reaction) found in complex biological matrices such as tissue, serum, urine, and other body fluids. These biomarkers provide a non-evasive means of clinical diagnosis. Accurate biomarker profiling of integrated clinical samples, however, has been difficult because of monitor response to interferring species causing erroneous analysis. Interferents in human biological samples can be endogeneous constituents, drugs, proteins, or metabolites, which respond at about the same wavelength as the biomarker of interest. Conventional compensatory methods have done little to improve accuracy and sensitivity. Thus, direct analysis of patient-derived samples has been limited.
One successful approach for proper analyses of biological samples is described in coassigned application Ser. No. 062,375, filed July 31, 1979 entitled "Method for Continuously Referenced Analysis of Reactive Components in Solution," now U.S. Pat. No. 4,263,406. A referenced, self-blanking system is disclosed therein for biomarker profiling of creatine kinase (CK,EC 2.7.3.2) and lactate dehydrogenase (LD,EC 1.1.1.27). Compensation for interferents is provided by a dual-channel flow system derived from a common source. Subtraction of a reference stream from a sample stream therein generates an indicator-species response correlatable to the profiled biomarker.
While self-referencing analysis produces reliable diagnostic assays, nonreferenced systems remain unreliable and of limited utility. Recently, the phenomena of ozone or peroxide-induced chemical luminescence have been described as a highly selective and extremely sensitive technique for analytical purposes.
Birks, T. W., et al., in "Chemical Luminescent Aerosol Spray Detector for Liquid Chromatography," Anal. Chem., 52, pp. 897-901 (1980) has quantitated highly fluorescent compounds by nebulizing a simple chromatographic effluent with a high velocity stream of O.sub.2 and O.sub.3 gases. Detection of the resultant ozone induced chemiluminescence has provided an accurate mass detection in the microgram to picogram range. The hazards of working with flammable concentrations of O.sub.2 /O.sub.3 gases and organic compounds are also discussed along with potential toxic wastes.
Another recent article has applied chemiluminescence to determine artificial samples of four dansylated amino acids. Kobayashi, S., et al., in "Determination of Fluorescent Compounds by High Performance Liquid Chromatography with Chemiluminescence Detection," Anal. Chem., 52, pp. 424-427 (1980) used laboratory reagents of hydrogen peroxide and oxalic esters to detect femtomole concentrations of the analyte. Authentic biological specimens were not therein analyzed.
Finally, Veazey, R. L., et al., in "Chemiluminescence High Performance Liquid Chromatographic Detector Applied to Ascorbic Acid Determinations," J. Chromatogr., 200, pp. 153-162 (1980) applied chemiluminescence detection to ascorbic acid and other organic reductants in basic media. The reactions involved are performed under optimal conditions and apply to any reducing agent. Only ascorbic acid was quantitated at truly useful concentrations. Reactant instability and variant flow rates necessitated frequent recalibration or addition of fresh reagents.